Electrospray ionization (“ESI”) is an important technique for the analysis of biological materials contained in solution by mass spectrometry. See, e.g., Cole, R. B. Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation & Applications; John Wiley and Sons, Inc.: New York, 1997. Electrospray ionization was developed in the late 1980s and was popularized by the work of Fenn. See, e.g., Fenn J B, Mann M, Meng C K, Wong S F & Whitehouse C M (1989), Electrospray ionization for mass-spectrometry of large biomolecules, Science 246, 64-71. Simplistically, electrospray ionization involves the use of electric fields to disperse a sample solution into charged droplets. Through subsequent evaporation of the droplets, analyte ions contained in the droplet are either field emitted from the droplet surface or the ions are desolvated resulting in gas phase analyte ions. The source of the liquid exposed to the electric field and to be dispersed is ideally one of small areal extent as the size of the electrospray emitter directly influences the size of droplets produced. Smaller droplets desolvate more rapidly and have fewer molecules present per droplet leading to greater ionization efficiencies. These ions can be characterized by a mass analyzer to determine the mass-to-charge ratio. Further analyte structural information can be obtained by employing tandem mass spectrometry techniques.
Separation of analytes prior to electrospray ionization is important for minimizing ionization suppression and MS spectral complexity. Microfluidic capillary electrophoresis with integrated electrospray ionization has been demonstrated as a fast and efficient method of coupling a liquid phase chemical separation with mass spectroscopy detection. See, e.g., Anal. Chem. 2008, 50, 6881-6887; and Anal. Chem. 2015, 87, 2264-2272. Conventional microfluidic methods that employ electrokinetic flow of sample into the separation channel are subject to injection bias and cannot effectively be used for some on-device sample focusing methods. Further, the injection of a well-defined band of sample into the separation channel of the microfluidic device can be important to achieving an efficient separation.
Most of the efforts to integrate sample processing with CE can be classified as either electrophoretic or chromatographic based. Electrophoretic based techniques, including sample stacking, sweeping, pH induced stacking, and transient isotachophoresis (tITP), can be simple to implement and can require little instrumentation development. Unfortunately, these techniques cannot typically load a sample volume larger than the volume of the capillary which limits the achievable concentration and sensitivity improvement. Furthermore, electrophoretic methods often concentrate matrix components equally to the analytes of interest, which can reduce separation performance. Finally, these methods can be limited to a narrow scope of analyte and buffer conditions, and may not be as widely applicable as other sample processing techniques.
On the other hand, chromatographic-based techniques, such as solid phase extraction (SPE), are typically more versatile than electrophoretic-based methods and can offer higher pre-concentration values based on the ability to load multiple capillary volumes onto the chromatographic sorbent. See, e.g., Ramautar et al. Electrophoresis 2014, 35, 128-137, the contents of which are hereby incorporated by reference herein. However, these methods present their own shortcomings. The presence of the SPE sorbent in the separation capillary can lead to clogging and disruption of the electroosmotic flow (EOF), reducing separation performance. Furthermore, in this scenario, matrix components enter the separation capillary, which can lead to wall interactions and further diminish the separation performance. On-line coupling, where the SPE sorbent is separate from the CE capillary but connected via a flow stream with tubing and valves, is the most common method for combining SPE with CE. The decoupling of the SPE sorbent from the CE capillary can inhibit or prevent clogging and EOF disruption. Additionally, the inclusion of valves between the SPE sorbent and the CE capillary can direct the matrix components to waste and prevent them from entering the CE capillary. Unfortunately, on-line coupling of SPE and CE often requires complex instrumentation. Furthermore, the transfer of the sample band from the SPE sorbent to the CE capillary typically introduces band broadening, limiting the resulting separation performance. Additionally, dead volume present in the on-line system can dilute the concentrated analyte band, reducing the amount of pre-concentration that can be achieved. Coupling sample processing with CE without sacrificing the separation performance can be a very challenging task.